Fig 2: Effect on proteins of the CDK4 and MDM2 pathway after CDK4/6 or MDM2 inhibition. The cells were treated with abemaciclib (Abe), ribociclib (Rib), and Nutlin-3a (N3a) at IC50 values for 72 h before protein extraction. This figure is a representation of the protein expression using different blots. The blots are delineated to clarify that belong to different gels or to different parts of the same gel. Each blot was normalized using KU80 as control. Exposure time for each protein analysis was the same for all cell lines.

Fig 3: Increasing HDAC1 rebalances histone acetylation and rescues proliferation and differentiation deficits of FMRP-deficient NPCs. a A schematic model showing that enhancing HDAC1 activities through either pharmacological or genetic reduction of MDM2 rebalances histone acetylation and rescues proliferation and differentiation of Fmr1 KO NPCs. b, c Western blot analyses of HDAC1 and FMRP protein levels in 6-mo NPCs treated with Nutlin-3 (n = 3). GAPDH was used as a loading control. d–f Western blot analyses of total and acetylated histone H3 (d, e) and histone H4 (d, f) in NPCs treated with Nutlin-3 (n = 3). Total histone H3 and total histone H4 were used as loading controls for acetylated H3 and H4, respectively. g, h Nutlin-3 treatment rescued the cell proliferation phenotype of Fmr1 KO NPCs as measured by BrdU incorporation (g, red; scale bar, 20 µm) followed by quantitative analysis (h, n = 3). i, j Nutlin-3 treatment rescued neuronal differentiation phenotypes of Fmr1 KO NPCs as assessed by Tuj1+ staining (i, red; scale bar, 20 µm) followed by quantitative analysis (j, n = 3). k, l Acute knockdown of MDM2 rescued the cell proliferation phenotype of Fmr1 KO NPCs as assessed by incorporation of the thymidine analog, BrdU (l, red, BrdU; green, GFP, scale bar, 20 µm) and quantitative analysis (l, n = 3). m, n Acute knockdown of EP300 rescued neuronal differentiation phenotypes of Fmr1 KO NPCs as assessed by a neuronal marker Tuj1+ (m, red, Tuj1; green, GFP, scale bar, 20 µm) followed by quantitative analysis (n, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, n.s.: no significant difference. Two-way ANOVA was used for all data analyses. Data are presented as mean ± s.e.m.

Fig 4: Scheme depicting our proposed Ras/NORE1A/ß-TrCP/MDM2 senescence mechanism. The interaction of NORE1A to ß-TrCP is Ras regulated, and this interaction regulates MDM2, a key negative regulator of p53 and Rb. Upon stimulation from Ras, MDM2 levels are suppressed by NORE1A and ß-TrCP allowing levels of p53 and Rb to rise and activate an oncogene induced senescence mechanism. Accordingly, loss of NORE1A removes this key MDM2 regulatory mechanism, allowing for levels of MDM2 to increase resulting in excessive down-regulation of powerful tumor suppressive mechanisms operated by p53 and Rb.

Fig 5: (A) HEK-293 cells transfected with expression constructs for MDM2 paired with an empty vector or a construct expressing NORE1A. MDM2 expression can be easily detected when co-expressed with an empty vector. Levels of MDM2 are suppressed when co-expressed with NORE1A; (B) HEK-293 cells were transfected with expression plasmids expressing NORE1A paired with either an empty vector or a construct expressing MDM2. 24 h post-transfection, the two groups were split and allowed to re-adhere to the culture dishes. Then the matched sets were treated with either DMSO (carrier) or MG132 at a final concentration of 10 µM for 12 h. The cells were lysed and examined on Western blot. Here we show that inhibition of the proteasome disrupts the ability of NORE1A to suppress MDM2, suggesting that indeed NORE1A is controlling MDM2 protein levels via the 26S proteasome. Representative blots of 2 independent experiments are shown in both panels.